MDMA electrochemical determination in aqueous media containing illicit drugs and validation of a voltammetric methodology - Core

Drug Analytical Research
 ISSN: 2527-2616 Drug Anal. Res., v. 4, n. 1, p. 3-11, 2020

 MDMA electrochemical determination in aqueous
 media containing illicit drugs and validation of a voltammetric methodology

Juliana Midori Toia Katayama a, Érica Naomi Oiye a,b, Maria Fernanda Muzetti Ribeiro a, Antônio José Ipólito c, José Fernando de
Andrade a, Marcelo Firmino de Oliveira a*

a
 Universidade de São Paulo – USP – Faculdade de Filosofia Ciências e Letras de Ribeirão Preto – Departamento de Química –
 14040-901 – Ribeirão Preto, São Paulo, Brazil
 b
 Henbak – 04710-160 – São Paulo – Brazil
 c
 SPTC – Núcleo de Perícias Criminalísticas de Ribeirão Preto – 14015-040 – Ribeirão Preto, São Paulo, Brazil

 *Corresponding author: marcelex@usp.br

MDMA is the abbreviation for 3,4-methylenedioxymethamphetamine, which is commonly found in “ecstasy” pills. The
psychoactive and euphoric effects that MDMA causes make this substance an illicit drug that is constantly seized by police forces.
We describe a low-cost and fast voltammetric methodology that requires a carbon paste electrode (working electrode) in aqueous
solution containing 0.1 mol L-1 LiClO4 as the supporting electrolyte. We conducted cyclic and square wave voltammetry and
obtained limits of detection of 0.33 μg mL-1 and 0.36 μg mL-1, respectively, as others figures of merit for a complete validation. It
includes the analysis main interfering substances, and results for seized samples were compared to those obtained by
chromatography, which were close. An extended study of robustness was carried out by Youden’s test, that is inedited to
electrochemical techniques when applied to forensic analysis. This test contributes to complete methodology validation and study
of electrode cost and efficiency during electrochemical measurements involving a carbon paste electrode. In the end, this work
presents a full validated methodology able to be applied in forensic laboratories.

Keywords: forensic chemistry, MDMA, voltammetry, validation.

 organizations, this group constantly updates their
Introduction recommendations to adapt to new equipment and drugs
 launched in the market [4].
The need for new sensorial experiences has become the main
reason why young people seek the various drugs that are For irrefutable results to be achieved, the SWGDRUG
available in the illegal market. Because hallucinogens and suggests that Infrared Spectroscopy, Mass Spectrometry,
amphetamines can distort visual and sensorial perception, Raman Spectroscopy, or Nuclear Magnetic Resonance
they are the most popular options. The hallucinogen Spectroscopy (group A) be combined with Liquid or Gas
amphetamine 3,4-methylenedioxymethamphetamine, Chromatography or Capillary Electrophoresis (group B) to
commonly known as MDMA, is found in ecstasy tablets, and identify the drug. If none of the group A methods is available,
it is one of the most consumed substances among synthetic two group B techniques can be combined with a group C test,
illicit drugs [1]. which encompasses colorimetric tests and tests that verify
 physicochemical properties, to certify drug identification [4-
MDMA stimulates the central nervous system: it creates 11].
empathogenic euphoria and elevates the user’s energy, but
this drug can also cause neurodegeneration. Severe Despite these recommendations, group A and B techniques
intoxication includes convulsions, coma, and hyperthermia are commonly expensive and require specific and highly pure
and can thus be fatal [2,3]. As a result of psychological reagents. This situation has encouraged researches to develop
disorders, MDMA users are also potential suicide victims and devices and methodologies that are cheaper and easier to
more susceptible to committing crimes and disturbing social operate, but which provide the same specificity as the other
peace. Therefore, these potential risks concern the local procedures [12-14].
authorities and call for efforts within the social, health, and
 Voltammetric methodologies can meet such requirements
police areas [1].
 and it might be adopted as routine analysis in forensic
For law enforcement, the police must properly identify the laboratories whose structure does not englobe equipment for
seized drug. Each country has their standard methods of drug techniques from class A. These methodologies demand
testing, and the recommendations of the Scientific Working simple apparatus and minimal use of reagents, and they offer
Group for the Analyses of Seized Drugs (SWGDRUG) are similar sensitivity to the sensitivity that is achieved with
frequently adopted. Created in 1997 by forensic scientists chromatographic methods. For example, a simple potentiostat
from the United States, England, Canada, Australia, Japan, may be achieved in small size, for portable analysis with a
Germany, the Netherlands, and several forensic

Drug Analytical Research
 ISSN: 2527-2616 Drug Anal. Res., v. 4, n. 1, p. 3-11, 2020

third of the budget for a gas chromatography. The analysis concentration of 1.0 mg mL-1. For the analysis of these
does not demand MilliQ water or high purity solvents. interfering substances solutions were prepared at a
 concentration of 5.0 μg mL-1 and analyzed in 3.0 mL of 0.1
Electroanalitycal chemistry presents several methods for mol L-1 LiClO4.
analysis of MDMA, cocaine, Δ9-tetrahydrocannabinol (Δ9-
THC), lysergic acid diethylamide (LSD), N-benzyl- The four ecstasy seized samples were also obtained from a
substituted phenethylamines (NBOMe), and other drugs [15- partnership between this research group and the same
29]. laboratory of toxicological analysis. A volume of 10-20 μL of
 a methanolic solution of these samples (1mg mL-1) was added
Cyclic voltammetry, differential pulse voltammetry, and to 3 mL aqueous solution of 0.1 mol L-1 LiClO4 and analyzed
square wave voltammetry are commonly applied to identify by cyclic and square ware voltammetry. The concentration of
drugs. Carbon paste as working electrode is an asset because MDMA in each sample was calculated by the linear equation
it is cheap and easy to handle. Graphite, carbon nanotubes, obtained by the respective analytical curve. The analysis was
and carbon black powder can be molded with an agglutinant performed in triplicate.
agent, like mineral oil or paraffin, resulting in an electrode
that can detect traces of seized drugs. Addition of a modifier Equipment
to the mixture can further improve electrode sensitivity and
selectivity [30-32]. All the measurements were performed on an µAutolab III
 potentiostat and on an Autolab PGSTAT128N
This work aims to employ both cyclic and square wave potentiostat/galvanostat operating with NOVA 1.11 software.
voltammetry with a carbon paste electrode to detect and to The last equipment was employed in the robustness test. The
quantify MDMA. The use of an aqueous medium following electrodes were used for the voltammetric
differentiates this methodology from chromatographic measurements: Ag/AgCl (containing saturated KCl solution)
analytical techniques, and the non-modified carbon paste as the reference electrode, a platinum wire (spiral or square)
electrode is an alternative to electrodes reported in the as the counter electrode, and a carbon paste electrode with
literature. This study focuses on analytical validation 10% paraffin (Sigma-Aldrich or Isogama) and 90% graphite
parameters and on Youden’s test as a different approach for power (Sigma Aldrich or Synth) in its composition as the
voltammetric procedures [2,16,33]. working electrode. After each measurement, the carbon paste
 electrode surface was renewed after being lightly slid on a
Material and methods sulfite paper.
Reagents and solutions Different paraffin brands were tested because they provided
 paraffin with distinct degrees of purity: Sigma-Aldrich
The performance of lithium perchlorate (LiClO4, Acros), supplies paraffin for laboratory purposes, whereas Isogama
sodium perchlorate (NaClO4, Vetec), potassium perchlorate supplies paraffin for handicraft purposes, like the production
(KClO4, Vetec), and ammonium perchlorate (NH4ClO4, of candles. The objective was to check how these two
Vetec) as supporting electrolyte was investigated. All the materials affected the voltammetric response.
solutions were prepared at a concentration of 0.1 mol L-1 in
distilled water. The pH variation of these supporting Voltammetric measurements were conducted within
electrolyte solutions was obtained using HCl or KOH, potentials ranging from -0.1 to 1.5 V. Pre-concentration was
depending on the chosen pH value. accomplished at -0.1 V for 10 s. Other pre-concentration
 times were also examined (5 s and 20 s). In Cyclic
The MDMA analytical standard solution was acquired from Voltammetry, the scan rates varied from 10 mV s-1 to 200 mV
Cerilliant and contained 1.0 mg of MDMA in 1.0 mL of s-1, to evaluate the electrochemical process nature.
methanol. Dilution of this standard was performed by the
addition of methanol (JT Barker), and successive aliquots of The Square Wave Voltammetry conditions were optimized in
2.5 μL of this standard solution, in concentration 1.0 mg mL- terms of frequency (5 to 40 Hz), amplitude (0.01 to 0.1 V),
1
 , were added to the electrochemical cell containing 3.0 mL and step potential (0.001 to 0.01 V). These parameters were
of 0.1 mol L-1 LiClO4 for the analytical curve. A blank set according to the lowest potential peak observed and
solution containing LiClO4 with 50 μL of methanol was also highest amperometric intensity.
analyzed.
 Specificity analysis against caffeine, cocaine, procaine,
For specificity analysis, 1.0 mg of the substances caffeine, lidocaine and theobromine was carried out in the same
procaine, lidocaine, and theobromine (Sigma Aldrich) was conditions and by using the same methodology as in the case
dissolved in 1.0 mL of methanol. The cocaine standard were of MDMA analysis.
obtained from a scientific partnership between this research
group and the laboratory of toxicological analysis – Institute The four ecstasy samples were also tested by High
of Criminalistics, Ribeirão Preto city, São Paulo state, Brazil Performance Liquid Chromatography, using an equipment
and was also dissolved in 1.0 mL of methanol. model Ultimate 3000 (Thermo Scientific) with a C8 column
 of Nano Science Technologies (25 cm x 4.6 mm, 5 μm),
The methamphetamine standard solution was acquired from coupled to a Diode Array Detector. A loop of 10 µL and flow
LGC in a concentration of 0.1 mg mL-1 and 3,4- rate of 1.2 mL min -1 was applied in a detector DAD. An
Metilenodioxianfetamine standard solution (LGC) was in a isocratic condition was used for the mobile phase, consisted

 4

Drug Analytical Research
 ISSN: 2527-2616 Drug Anal. Res., v. 4, n. 1, p. 3-11, 2020

of 85 % de phosphoric acid (0.5 % v/v) + triethylamine (for when combination 1 is tested, the result will be “s”; when
pH adjustment 2.35) and 15 % acetonitrile. For the analytical combination 2 is tested, the result will be “t”; and so on, until
curve was used the range concentration of 5.0 μg mL-1 to 100 all the eight combinations have been tested.
μg mL-1 of MDMA. The samples solutions were prepared
with concentration of 0.1 mg mL-1. The applied methodology In each combination, MDMA solution analysis was carried
is based on the validated method proposed by UNODC [34]. out in triplicate, at the work concentration (1.99 μg mL-1). The
The analysis was performed in triplicate. result observed in each combination was the current (A) in
 two voltammetric techniques (Cyclic and Square Wave
Validation and Robustness evaluation (Youden’s test) Voltammetry).
For the MDMA measurement validation process, the figures To determine the influence of a factor, the four values
of merit such as the Limit of Detection and the Limit of corresponding to the capital letters (nominal conditions) and
Quantification were calculated as 3*ơ/m and 10* ơ/m the four values corresponding to the lowercase letters
respectively, being ơ the standard deviation observed in the (variation) had to be found, and the means of the these two
linear fit for the linear coefficient and m the voltammetric groups had to be compared. For example, to calculate how the
sensitivity. potentiostat (A/a) affected the final results, Equation 1 was
 employed:
In this study, reproducibility was determined as being the
deviation observed on different days of analysis, whilst + + + w+x+y+z
 Effect A/a = 4
 - 4
 Eq. 1
repeatability was calculated on the same day of analysis, with
electrode surface renewal: a total of seven assays were All the seven pairs were determined to obtain seven effects,
performed for each of these parameters, to determine the which were ordered to reveal which experimental parameters
standard deviation. significantly impacted the result of analyses.
The voltammetric method robustness for MDMA Results and Discussion
quantitation was evaluated by using the method proposed by
Youden and Steiner (1975) and it implies in studying the most First, we investigated the use of perchlorate salts, such as
impacting factor in analysis. The Younden’s test is found in lithium, sodium, potassium, and ammonium perchlorates as
recommendations for validation test in guidelines such as supporting electrolyte for MDMA voltammetric detection.
AOAC [35,36]. Eight separate experiments were conducted We chose the perchlorate anion because supporting
to determine how the seven selected parameters influenced electrolytes bearing this anion have been commonly reported
the system. Table 1 lists the applied experimental parameters in works on the electrochemical detection of synthetic drugs
and the nominal values. [17,23-25]. The voltammetric response of LiClO4 exhibited
 an electric current peak of greater intensity, followed by
Table 1. Experimental parameters and variations for robustness
 NaClO4, KClO4, and NH4ClO4, as indicated in Figure 1. Thus,
evaluation.
 by comparing the perchlorate salts, we were able to associate
 Nominal the voltammetric response with the cation size: the smaller
 Parameter Variation the cation, the higher the peak intensity. The results were
 condition
 Potentiostat µAutolab Autolab similar for both Cyclic Voltammetry and Square Wave
 A/a A a Voltammetry, and the future measurements were taken with
 model III 128N
 Counter LiClO4 in aqueous medium.
 B/b electrode Spiral B Square b
 format
 Sigma
 Graphite Synth
 C/c C Aldrich c
 supplier (99.0%)
 (99.99%)
 Sigma
 Isogama Aldrich
 Paraffin
 D/d (unknown D (for d
 supplier
 purity) laboratory
 purpose)
 E/e Light Absence E Presence e
 N2 Flow
 F/f 0 F 10 F
 time (s)
 Quality of H2O H2O
 G/g G G Figure 1. Voltammetric response for MDMA (1.66 μg mL-
 water distilled MilliQ
 1) in different aqueous supporting electrolyte solutions (for
 KClO4 and NaClO4: frequency = 25 Hz, amplitude = 0.05 V,
The eight voltammetric runs were randomly accomplished. and step potential = 0.005 V; for NH4ClO4, LiClO4, and LiCl:
Table 2 summarizes the factorial combination of the seven frequency = 35 Hz, amplitude = 0.07 V, and step potential =
parameters and their respective variations for Youden’s test; 0.005 V; pre-concentration was conducted at -0.1 V for 10 s
the results of the determinations are shown from s to z. Hence, in all the measurements).
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The anodic peak observed for MDMA at 1.24 V for cyclic
voltammetry and 1.20 V for square wave voltammetry.
 Table 2. Factorial combination of the experimental parameters for robustness evaluation by Youden’s test.
 Parameter Experiment number
 1 2 3 4 5 6 7 8
 A/a A A A A a a a a
 B/b B B b b B B b b
 C/c C c C c C c C c
 D/d D D d d d d D D
 E/e E e E e e E e E
 F/f F f f F F f f F
 G/g G g g G g G G g
 Observed results S t u v w x y z

We checked the MDMA oxidation signal behavior in the As the scan rate increases, it is possible to observe the
LiClO4 solution at pH 2.0, 5.0 and 7.0. As reported before, displacement of the oxidation peaks to more positive
there is a dependence between oxidation peak and pH, with potentials, which suggests the irreversible nature of the
an increment in pH the potential peaked decreases and the oxidation process [38]. We decided to employ a scan rate of
current increase [15]. At pH 2, a low peak current was 50 mV s-1 during the cyclic voltammetry experiments.
observed, which increased with increasing pH to 5. Basic
medium also becomes easy the oxidation of primary and The dependence of ip versus v-1/2 on v was checked for
secondary amines, as in the case of MDA and MDMA oxidation. The current function (ip versus v-1/2) is
methamphetamine, that are considered MDMA interfering independent of the scan rate for reversible and irreversible
substances [15]. To avoid this interference, pH 5 was chosen processes [38]. In the case of MDMA oxidation, the decrease
for voltammetric analyzes of MDMA. of the current function with v indicates that a chemical
 reaction is coupled to the electrode process and characterized
The potential scan rate was varied between 10 and 200 mV s- an EC mechanism [38].
1 (Figure 2). Through the relation between scan rate and
current values, we can obtain conclusions about the kinetics The anodic peak observed can be related to an oxidation of
and mechanism of the reaction involved. The linearity the aromatic nucleus of the MDMA molecule. Figure 3 shows
between the peak current and the square root of scan rate a proposed electro-oxidation and formation of a cation radical
characterizes a process of diffusion of the analyte to the of MDMA in aqueous media based on literature [7,15,33].
electrode surface [38]. The log ip vs. log v curve is linear with Garrido et al. [15] associated the first anodic peak with that
slope of 0.36 and also indicates a diffusion-controlled cation radical formation and a second and third oxidation
electrode process. A slope close to 0.5 is expected for peak to a dimerization process of these radicals followed by
controlled-diffusion electrode processes and close to 1.0 for the oxidation of the secondary amine present in MDMA
controlled- adsorption electrode processes [38]. molecule. In this studied conditions we observed only the first
 oxidation peak.

 -6
 2.8x10
 -1
 10 mV s
 -6
 1.8x10
 -6
 1.6x10
 -6 -1
 2.4x10 25 mV s -6
 1.4x10
 -6
 -1 1.2x10
 50 mV s
 i (A)

 -6
 1.0x10
 -6
 2.0x10 75 mV s
 -1 -7
 8.0x10
 -7
 6.0x10
 -1
 100 mV s -7
 4.0x10
Current (A)

 -6
 1.6x10 -1
 2 4 6
 1/2
 v
 8 10
 1/2
 (mV s )
 -1/2
 12 14 16

 200 mV s
 -6
 1.2x10 Figure 3. Mechanism for electro-oxidation of MDMA in
 -7
 aqueous media [7,15,33].
 8.0x10

 4.0x10
 -7 Validation

 0.0 Once we had performed the optimization and verified the
 experimental conditions, we obtained important information
 1.0 1.1 1.2 1.3 1.4
 for the validation and the possible quantification of MDMA
 Potential (V) vs. Ag/AgCl
 from the analytical curve. Table 3 presents the Cyclic
 Voltammetry and Square Wave Voltammetry results
Figure 2. Voltammetric response of MDMA (2.59 μg mL-1) obtained after successive additions of the standard solution to
at different scan rates. The supporting electrolyte was LiClO4 the electrochemical cell, as well as the parameters for the
at 0.1 mol L-1 (pre-concentration at -0.1 V for 10 s).
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equation, which described the linear relation between the The limit of detection obtained for Cyclic Voltammetry and
current and the presence of MDMA. We obtained the data Square Wave Voltammetry, respectively, corresponded to the
from the peak current illustrated in Figure 4. presence of 0.33 µg and 0.36 µg of MDMA in a tablet or pill
 dissolved in 1.0 mL of methanol. This value was lower than
Table 3. Analytical parameters calculated for MDMA the values found in seized samples, more than 50 mg per pill
analysis by Cyclic Voltammetry and Square Wave [2].
Voltammetry.
 The techniques afforded close values of linearity,
 Cyclic Square Wave reproducibility, repeatability, and linear range. In general,
 Parameter
 Voltammetry Voltammetry Cyclic Voltammetry provided more satisfactory accuracy and
 Limit of Detection 0.33 μg mL-1 0.36 μg mL-1 limits of detection and quantification, as the values were
 Limit of Quantification 1.11 μg mL-1 1.22 μg mL-1 lower than those obtained by Square Wave Voltammetry
 1.11 to 4.97 μg 1.22 to 4.97 applied to MDMA analysis in this work.
 Linear range
 mL-1 μg mL-1
 Linearity 0.9941 0.9929 As explained before, robustness evaluation (Youden’s test)
 fixed the same MDMA concentration, so we were able to
 Accuracy 98.32% 95.26%
 determine which factor influenced the analysis the most at the
 3.78 10-2 A L 2.64 10-1 A L
 Sensitivity (slope) end of the eight assays. Table 4 reports the amperometric
 mol-1 mol-1
 responses for each assay.
 Linear coefficient -7.45 10-8 A 1.75 10-6 A
 Reproducibility Table 4. Current values obtained in eight runs performed for
 3.06% 3.80%
 (intra-day precision) Youden’s test (MDMA concentration of 1.99 μg mL-1).
 Repeatability
 3.23% 3.62% Cyclic Square wave
 (inter-day precision) SD SD
 Experiment Voltammetry Voltammetry
 (%) (%)
 (A) (A)
 1 5.26 10-7 1.42 4.84 10-6 1.34
 2 4.88 10-7 1.23 5.15 10-6 0.89
 3 4.9110-7 0.98 5.02 10-6 1.46
 4 4.90 10-7 1.67 5.16 10-6 2.66
 5 5.17 10-7 1.40 4.82 10-6 0.65
 6 5.45 10-7 1.30 5.28 10-6 2.82
 7 4.91 10-7 4.00 5.09 10-6 1.42
 8 5.26 10-7 1.14 4.79 10-6 2.88

 The results in Table 4 show the proximity between the peak
 current values in all the eight experiments. Table 5 lists the
 values obtained for each factor after we applied Equation 1.
 On the basis of these values, we were able to compare the
 influence of each parameter numerically.
 Table 5. Effects of the robustness evaluation proposed by
 Youden’s test.

 Cyclic Square wave
 Effect
 Voltammetry Voltammetry
 A/a -2.111 10-8 4.825 10-8
 B/b 1.929 10-8 5.250 10-9
 C/c -5.993 10-9 -1.513 10-7
 D/d -2.692 10-9 -1.023 10-7
 E/e 2.549 10-8 -6.875 10-8
 F/f 1.129 10-8 -2.340 10-7
 G/g 7.907 10-9 1.487 10-7

 The presence or absence of light was chosen as one of the
Figure 4. Voltammograms obtained for successive MDMA factors in the Youden test because MDMA standards are
concentrations, in LiClO4 as supporting electrolyte: A) Cyclic purchased in light-protected ampoules. We want to check if
Voltammetry (pre-concentration at -0.1 V for 10s, scan rate = the light affects the voltammetric analysis of MDMA. This
50 mV s-1); B) Square Wave Voltammetry (frequency = 35 factor (E) was the one that most affected the analysis in Cyclic
Hz, amplitude = 0.07 V, step potential = 0.005 V; pre- Voltammetry, followed by factor F (nitrogen flow) in Square
concentration at -0.1 V for 10 s). Wave Voltammetry.
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The application of nitrogen flow is a common procedure in MDMA has a secondary and MDA a primary amine group
voltammetric measurements to remove electroactive oxygen, [15]. A better differentiation between these two molecules
and it is applied before analysis. Once this step is not would require the use of a chromatographic technique.
necessary, it turns the methodology simpler and faster, as it
implies in removing a step in experimental procedure and it
also does not imply in acquiring other chemical supply for
this nitrogen.
The negative value indicated that factor variance (nitrogen
flow time established as 10 s) was the most decisive
parameter in Square Wave Voltammetry. However, an
additional experimental step pointed to a small difference in
peak current. This was based on the average peak current
calculated for experiments 2, 3, 6, and 7, where the nitrogen
flow was applied: there was a difference of around 0.3 10-6 A
as compared to the experiments carried out without nitrogen
flow (1, 4, 5, and 8).
The most important information obtained during robustness
evaluation was the possibility of reducing the costs inherent
to this voltammetric analysis: the method did not require
high-purity graphite; indeed, a simpler powder provided
similar responses. The use of a paraffin from candle
decreases the total cost of the analysis, as it is not necessary
affording a commercial one with higher purity from chemical
industry.
Youden’s test reinforced the possibility of using distilled
water to prepare the supporting electrolyte solution, which
simplified the methodology as MilliQ water production was
unnecessary. This is not possible in the case of
chromatographic equipment.
This robustness evaluation therefore completely validated the
analysis, which requires a cheaper electrode for MDMA
detection.
Interfering substance analysis
Because some substances may be added to ecstasy Figure 5. Cyclic voltammograms of A) caffeine, cocaine,
tablets/pills or even substitute MDMA present in them, we lidocaine, theobromine, metamphetamine and MDMA (5.00
also examined the voltammetric response for the standard µg mL-1); B) procaine, MDA and MDMA (5.00 µg mL-1).
solution of caffeine, cocaine, procaine, lidocaine, Scan rate: 50 mV s-1 and pre-concentration at -0.1 V for 10s.
theobromine, methamphetamine and 3,4-
Methylenedioxanphetamine (MDA) in the presence or in the Analysis of samples
absence of MDMA. Only procaine and MDA had an We used the chromatographic and electrochemical
amperometric response, as the MDMA, Figure 5 (A). methodologies to analyze the four seized ecstasy pills. For
The procaine displayed an anodic peak at approximately 1.04 both techniques, an analytical curve was used to quantify the
V for Cyclic Voltammetry and 1.02 V for Square Wave samples, and Table 6 compiles these results for MDMA
Voltammetry [37], therefore, we conducted a study in determination in ecstasy pills by HPLC, Cyclic Voltammetry,
medium also containing MDMA, as depicted in Figure 5 (B). or Square Wave Voltammetry, performed in triplicate for
Nevertheless, MDMA peak (2) shifted to higher potential each sample. The calculated values of MDMA in ecstasy
(1.26 V) for both techniques, but this did not prevent both samples 2 and 4 were close to the values achieved with the
drugs from being detected in the same matrix. Hence, the chromatographic technique. However, the values obtained for
present methodology is specific for MDMA analysis even in samples 1 and 3 in the voltammetric technique showed a
the presence of its main interfering substances. considerable difference. Thus, it is suggested that the
 methodology developed may assist in the presumptive
The MDA presented a peak at 1.24 V for Cyclic Voltammetry analysis of MDMA. We found between 58 and 93 mg of
and 1.19 V for Square Wave Voltammetry, very close to MDMA in ecstasy pills (considering a total mass of 250 mg)
MDMA oxidation peak. Because MDA is an MDMA and the typical dosage of MDMA for recreational use varies
metabolite, they have a very similar structure. The only from 50 mg to 150 mg [39]. Low dose (between 50 and 75
structural difference between them lies in the amine group, mg) used on single occasion produced the desired effects by
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most users like euphoria, well-being, sharpened sensory moderate MDMA use: A systematic review. Neurosci.
perception and sociability. At higher doses, undesired effects Biobeha. Rev. 2016, 62, 21–34.
may appear like headache, nausea, loss of appetite, blurred
vision, insomnia, panic attacks, delirium or even brief 3. De la Torre R, Farré M, Roset PN, Pizarro N, Abanades
psychotic episodes [40]. S, Segura M, Segura J, Camí J. Human pharmacology of
 MDMA pharmacokinetics, metabolism, and disposition.
Table 6. MDMA quantification in seized ecstasy pills. Ther. Drug Monit. 2014, 26, 137-144.
 Cyclic Square Wave
 HPLC 4. SWGDRUG, Scientific working group for the analysis of
 Sample Voltammetry Voltammetry
 (% m/m) sized drugs (swgdrug) recommendations (2016)
 (% m/m) (% m/m)
 1 25,44 ± 1,65 28,85 ± 1,13 20,46 ± 0,62 Washington,
 2 37,39 ± 3,92 35,53 ± 2,68 34,44 ± 1,30 http://www.swgdrug.org/Documents/SWGDRUG%20Re
 3 23,22 ± 5,12 28,32 ± 1,39 20,33 ± 1,21 commendations%20Version%207-1.htm. Accessed in 16
 4 28,63 ± 3,72 27,45 ± 5,99 26,06 ± 2,79 august 2019

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 Analysis of illicit drugs by nonaqueous capillary
 electrophoresis and electrochemical detection. Fresenius
We have demonstrated new methodologies for MDMA J. Anal. Chem. 2000, 367, 359-363.
determination by Cyclic Voltammetry and Square Wave
Voltammetry, in aqueous medium. Both techniques provided
 6. Alizadeh N, Mohammadi A, Tabrizchi M. Rapid
close values of figures of merit, but Cyclic Voltammetry
 screening of methamphetamines in human serum by
afforded more satisfactory values of limits of detection and
 headspace solid-phase microextraction using a
quantification. Robustness evaluation helped to establish the
 dodecylsulfate-doped polypyrrole film coupled to ion
complete validation process and to prove whether it is
 mobility spectrometry. J. Chromatogr. A. 2008, 1183, 21-
feasible to apply the methodology to analyze seized samples
 28.
in forensic laboratories on a routine basis. The methodologies
allow for simple, fast, and sensitive MDMA analysis with a
cheap working electrode. They are also specific for MDMA 7. Milhazes N, Martins P, Uriarte E, Garrido J, Calheiros R,
analysis even in the presence of its main interfering Marques MPM, Borges F. Electrochemical and
substances like caffeine, cocaine, procaine, lidocaine, spectroscopic characterization of amphetamine-like
theobromine and methamphetamine. The same methodology drugs: Application to the screening of 3,4-
can distinguish between MDMA and procaine, which allows methylenedioxymethamphetamine (MDMA) and its
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Acknowledgments 8. da Silva DG, de Pinho PG, Pontes H, Ferreira L, Branco
 P, Remião F, Carvalho F, Bastos ML, Carmo H. Gas
The authors acknowledge the financial support of Conselho chromatography–ion trap mass spectrometry method for
Nacional de Desenvolvimento Científico e Tecnológico the simultaneous measurement of MDMA (ecstasy) and
(CNPq), Fundação de Amparo à Pesquisa do Estado de São its metabolites, MDA, HMA, and HMMA in plasma and
Paulo (FAPESP - Process 2016/23825-3), and Coordenação urine. J. Chromatogr. B Analyt. Technol. Biomed. Life.
de Aperfeiçoamento de Pessoal de Nível Superior (Process Sci. 2010, 878, 815-822.
Capes Pro Forenses 25/2014). Finally, we would like to thank
Dr. Cynthia Maria de Campos Prado Manso for revising and 9. Barnes AJ, Scheidweiler KB, Kolbrich-Spargo EA,
editing the text. Gorelick DA, Goodwin RS, Huestis MA. MDMA and
 metabolite disposition in expectorated oral fluid following
 controlled oral MDMA administration. Ther. Drug Monit.
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